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Introduction Broth testing methods Macrodilution Microdilution Agar dilution Automated Antimicrobial Susceptibility Testing (AST) systems Vitek-2 Phoenix Microscan Sensititre Considerations in evaluating AST systems
Introduction
Various methods of antibiotic susceptibility testing are: 1. Quantitative Methods 2. Qualitative Methods 3. Automated Susceptibility Tests 4. Newer Non-Automated Susceptibility Tests 5. Molecular Techniques
Broth preparation Antimicrobial agent Reference methods for in vitro susceptibility testing 1. Macrobroth dilution (Tube dilution) 2. Microbroth dilution
- Stock solution - Working solutions
Preparation of tubes/ plates Inoculum preparation Inoculation of tubes/ plates Incubation of tubes/ plates
Broth preparation MuellerHinton broth (MHB) General purpose medium (non fastidious) Cation- adjusted Optimum pH
-Appropriate solvent for dilution (CLSI M100 Table 5A) -Stock solutions may be stored at -60C for more than 6 months for most antimicrobial agents -Avoid repeated freeze thaw
Inoculum preparation Working solution - Serial doubling dilutions - Dilution scheme available in CLSI M100 Direct colony suspension method Most convenient Fresh colonies; 0.5 McFarland standard Fastidious organisms (eg: Neisseria gonorrhoeae; Haemophilus spp)
Growth method - When smooth suspension cannot be made - Non- fastidious organisms
Incubation of tubes/ plates -Incubate inoculated macrodilution tubes/ microdilution trays at 35 +/- 2C for 16- 20 hours in ambient air incubator -Within 15 minutes of adding inoculum -Do not stack trays >4 high -Seal each tray
https://www.boundless.com/image/measuring-minimal-inhibitoryconcentration-via-the-microbroth-dilution-method/
http://archive.ispub.com/journal/the-internet-journal-of-herbal-and-plantmedicine/volume-1-number-1/the-antibacterial-or-antifungal-effects-ofeurycoma-longifolia-root-extract.html
Reading results QC passed; GC wells + The MIC is the lowest concentration of the agent that completely inhibits visible growth as judged by the naked eye
Quality control To include at least 1 control organism (ATCC) with each batch of testing Test values for the control strains should be within the published range
Limitations
Labour intensive Strict adherence to protocol is required The MIC value is not the sole predictor for clinical outcome
Antimicrobial agent
- Stock solution - Working solutions
Preparation of agar and plates Inoculum preparation Inoculation of plates Incubation of plates Reading results
Quality control
Agar and Plates preparation MuellerHinton agar is considered the reference medium One concentration of antibiotic/ plate Include a drug free control
Allow the sterilized agar to cool to 50C in a water-bath, before adding antibiotics Set at room temperature. Do not overdry. Store plates at 4-8C
Inoculation of plates Mark the plates for orientation Use an inoculum- replicating apparatus to transfer the inocula to the series of agar plates Allow the inoculum spots to dry at room temperature before inverting the plates for incubation.
Reading results
Limitations
Labour intensive Strict adherence to protocol is required The MIC value is not the sole predictor for clinical outcome
Several automated systems for antimicrobial susceptibility testing are commercially available
Examples:
- Vitek 2 System (Bi oMrieux) - Phoenix Automated Microbiology System (SD Di agnostic System) - MicroScan Walkaway System (Si emens Healthcare Diagnostics) - Sensititre Aris 2X (Trek Di agnostic System)
General advantages
Spa ce Qua ntitative results (MIC va lues) Reproducibility Cos t- effective for laboratories with high throughput Reduction i n labour Ea s e of performance
General limitations
Cos t Regular maintenance required with upgrading of computer s oftware La g ti me i n upgrading of new breakpoints i n s oftware Ma nual preparation of inoculum Li mi ted ra nge of organisms Li mi ted a ccuracy i n certain organism-antimicrobial combi nations Li mi ted flexibility i n antibiotic panels Tes ting space on the antibiotic s usceptibility ca rds is not i nfinite, a nd therefore not a ll MICs ca n be tested (eg Test ra nge MIC 2 g/ml ) No mi xed culture
Fa ster reporting of s usceptibility results Convenient i nterface with the laboratory i nformation system (LIS) Expert s ys tems software to i nterpret s usceptibility results involving a typi cal patterns a nd unusual resistance phenotypes
Vitek 2 Principle:
Utilized growth-based technology Uses compact colorimetric reagent cards that are incubated and interpreted automatically
Phoenix
Principle: This system uses an optimized colorimetric oxidation-reduction indicator to detect organism growth in the presence of an antimicrobial agent for susceptibility testing
http://www.biomerieux-industry.com/servlet/srt/bio/industrymicrobiology/dynPage?open=NDY_IND_BPA_PRD&doc=NDY_BPA_PRD_G_PRD_NDY_10&pubparams.sform=4&lang=en
MicroScan WalkAway
Principle: Large self-contained incubator/reader device that can incubate and analyze 40-96 microdilution trays with a photometer/ fluorometer to determine growth development
Sensititre Aris 2X
Principle: Automated, overnight, incubator/reader device that can incubate and analyze up to 64 microdilution trays. Growth is determined by fluorescence measurement after 1824 h of incubation.
Thank you
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